Analytical method development and validation are one of the very imp aspects in Drug testing and approval process.Here I tried to explain the same with my experience.

1. By
Sada Siva,Scientist,
Analytical Development,
Aurobindo Pharma Ltd (Biologics) .
Analytical Method Validation

2. Analytical Method life cycle
4.Method Validation
To prove method has the ability to
perform for its intendted use
complete Documentation
3. Method Transfer to QC
Method transfer to end user /clients
5. Periodically Monitoring / Review
of Methods in Control Labs
1. Analytical method development
To optimize a method for intended use
2. Qualification of method
To prove method has the ability to
perform for its intended use
Intial Documentation
6. Method Revalidation
If any changes in method / method
perfomance changes with time

3. Validation-Introduction
 What
 Why
 Where
 Who
 When
 How
 Requirements
 Case study

4. What is a validation?
 It is a process of demonstrating that method is
suitable their intended use with documentation.
 Validation should verify the suitability of an analytical
method for its intended purpose
 Validation may be performed in different ways
(individual purpose) according to common
standards/guidelines

5. Why do we need to do validation?
Regulatory bodies require safety and quality information
of our product for approvals.
For this one should need to have reliable test methods.
That reliability of method is provided by doing a
validation exercise ., and which is documented.
*It is also essential part of GMP practices

6. Where , when & who will perform
Validation ?
 Where:
Generally validation is performed in a GxP laboratory
or Equivalent.
mostly it should be performed in End user (QC)
laboratory where the method going to be used
regularly.
 Who:
A qualified analyst (GxP trained, qualified and skilled )
 When:
Before commercialization (during CT3 time-
biosimilars )
Before CT or IND application ( for New Drugs)

7. Pre-requisites of validation?
 Prerequisites for analytical method validation
 Six “M”s
Ready to start
Validation
Man Machine
qualified
calibrated
robust
qualified
Methods
suitable
characterised
documented
MilieuMaterial Management
Quality
Reference
standards
Tempe-
rature
Analysts´
support
skilled
Humidity
Vibrations Time
Supplies
Irradi-
ations

8. How to perform validation?
 Many regulatory bodies have guideline how to perform
validation of analytical method.
 Some of them are ICH Q2, FDA, USP, EMEA

9. Validation characteristics (parameters)
 Accuracy
 Specificity
 Precision
Repeatability
Intermediate precision
Reproducibility
 Linearity
 Limit of detection
 Limit of quantitation
 Range
 Robustness

10. Degree of validation depends on Test

11. System suitability and Acceptance
criteria
 Integral part of each method/procedure to ensure that
all analytical operations, electronics and equipment's
are working properly at the time of analysis.
 Appropriate system suitability and its acceptance
criteria should be defined.
 Resolution ,tailing factor, RRT , capacity factor, no . of
theoretical plates between 2 peaks in chromatography
methods
 Minimum height /response of control samples
 Spike recovery in sample processing (extraction) is there.
 BC recovery of known amount of control sample
 In every parameter it should comply or pass if not the test
will be considered (not used)

12. Specificity
 Specificity Is the ability to assess unequivocally the
analyte in the presence of components which may be
expected to be present (impurities, degradants,
matrix…)
Identity testing
 To ensure the identity of an analyte
Purity testing
 To ensure accurate statement on the content of
impurities of an analyte
Assay
 To allow an accurate statement on the content of an
analyte in a sample

13. Specificity-Approaches
 Selectivity: the extent to which a method can determine
particular analyses in mixtures in matrix without interference
from other compounds (used in chemistry but in specificity
is the ultimate of selectivity-IUPAC)
SAMPLE PURPOSE
SYSTEM SUITABILITY SAMPLE SYSTEM SUITABILITY
FORMULATION BUFFER (SEC,CEX)
TO EVALUATE MATRIX
INTERFERENCE OR
TO EVALUATE
SEPARATION
SELECTIVITY
TEST SAMPLE DILUTED IN FB, WFI (OD 280,SEC)
TEST SAMPLE
TEST SAMPLE SPIKED WITH IMPURITY (HCD)
IMPURITY SAMPLE NEAT (HCP,HCD)
UNRELATED SAMPLES (GLYCAN, SDS-
PAGE,HCD)
SYSTEM SUITABILITY SAMPLE SYSTEM SUITABILITY

14. Precision
• Expresses the closeness of agreement between a
series of measurements (replicates) obtained from
multiple sampling(different vials) of the same
homogenous sample (same batch)
• Is usually expressed as the standard deviation
(S), variance (S2) or relative standard deviation
(RSD) of a series of measurements
• Precision may be considered at three levels
 Repeatability (intra-assay precision)
 Intermediate Precision (variability within a laboratory)
 Reproducibility (precision between laboratories)

15. Repeatability-Approaches
 Repeating the analysis under same operating conditions in a short
time interval
 Same samples analysis(different vials from same batch) in same
sequence.
 Independent sample preparations (6) will be done but analysed in a
single assay.
Sample Name HMW Peak
RT Area % of Area
System suitability sample 3.24 58481 0.88
Test sample Replicate 1 3.24 109382 1.59
Test sample Replicate 2 3.24 109560 1.58
Test sample Replicate 3 3.24 110185 1.58
Test sample Replicate 4 3.24 108539 1.58
Test sample Replicate 5 3.24 109996 1.59
Test sample Replicate 6 3.24 110362 1.59
Test sample con 1(triplicates)
concentration based precision (3 replicate / 3
different concentration)
Test sample con 2 (triplicates)
Test sample con 3 (triplicates)
%RSD 0.00 1 0.17

16. Intermediate Precision-
Approaches
 Same test sample is analysed varying any one
test condition like analyst , Day, Instrument,
reagents/consumables of different lots etc.
Sample Name HMW Impurity
RT Area % of Area
Test sample _Ctrl 3.24 109670 1.58
Test sample_Inter instrument 3.24 109851 1.31
Test sample_Inter analyst 3.24 120053 1.49
Test sample_Interday 3.24 116089 1.53
P2_Test sample_Inter column 3.26 113579 1.57
%RSD 0.21 4 7.39

17. Reproducibility
 Precision between two laboratories.
 Interlab precision.
 Generally done in labs working with collaboration /
pharmacopeia methods
 Generally not required in industries.
 Ruggedness (USP) is defined as the degree of
reproducibility of results under variety of conditions like
different labs, Environmental conditions, materials ,
operators, instruments and analysts.
Robustness – deliberate changes in operations conditions
Ruggedness-normal , expected operations conditions (many
labs)
Reproducibility- normal operations conditions in 2 or 3 labs
(clients)

18. Accuracy
• Expresses the closeness of agreement between
the value which is accepted either as a
conventional true value (Expected) or an
accepted reference value and the value found
(Observed value by method)
 Sometimes referred to as „TRUENESS“/ Sameness
 Agreement of test results with the true value
 Expressed in % Recovery

19. Accuracy- Approaches
 Spiking known amount in sample (HCD,SEC) and
calculating the spike recovery (3 conc /3 replicates)
 Spiking of ref.mateterial in placebo.
 Known amount (purity) of sample is taken and
analysed to check method recovery(80-120%
acceptable range )
sample Expected Observed % Recovery
sample-1 100 98 98
sample-2 80 81 101
sample-3 20 19 95

20. Precision and Accuracy
Accurate &
precise Accurate &
imprecise Inaccurate &
precise Inaccurate &
imprecise

21. Linearity
 Linearity of an analytical procedure is its ability (within a
given range) to obtain test results which are directly
proportional to the concentration (amount) of analyte in
the sample
 If there is a linear relationship, test results should be
evaluated by appropriate statistical methods
 Correlation coefficient (r)
 Y-intercept
 Slope of regression line
 Residual sum of squares (R2)
 Plot of recovey

22. Linearity Approaches
 Different con (minimum 5) of sample Vs responses
(PAHPLC,OD 280,HCD)
 Spiking of known amount of impurity/substances into
test sample (SEC ,CEX, Glycan)

23. Range
 Range
The range of an analytical procedure is the interval between the
upper and lower concentration (amounts) of analyte in the sample
for which it has been demonstrated that the analytical procedure
has a suitable level of precision, accuracy and linearity

24. Range-Approaches
 Data from linearity/accuracy is used to check this.
 Have to check how method behaves in different ranges like
Ex: Complete range of method used in linearity (0.1 to 120 %)
Lower range of linearity/accuracy samples (0.1 to 10%)
Middle range of linearity/accuracy samples (10 to 100%)
Higher range of linearity/accuracy samples (50-120%)

25. Limit of Quantitation (LOQ, QL)
 Limit of Quantitation (LOQ, QL)
 The LOQ is the lowest amount of analyte in a sample
which can be quantitatively determined with suitable
precision and accurac
 Determination is usually based on
 Signal to noise ratio (~10:1) (baseline noise)
-- Standard deviation of response (s) and Slope (S)
 Viaual evaluation
 Calibration curve
 SD of blanks

26. Limit of Detection (LOD, DL)
 Limit of Detection (LOD, DL)
 The LOD of an analytical procedure is the lowest
amount of analyte in sample which can be detected
but not necessarily quantitated as an exact value
 Determination is usually based on
 Signal to noise ratio (~3:1) (baseline noise)
 Standard deviation of response (s) and Slope (S)
 Viaual evaluation
 Calibration curve
 SD of blanks

27. LOD and LOQ calculations

28. Robustness
 Robustness
 Robustness of an analytical procedure should
show the reliability of an analysis with respect to
deliberate variations in method parameters
 The evaluation of robustness should be
considered during the development phase

29. Robustness-Approaches
 Deliberate changes like reagents/buffers pH,
concentration, temperature, injection volume,
Incubation time , Samples stability, pippete errors
etc depending on type of methods
If measurements are susceptible to variations in
analytical conditions the analytical conditions
should be suitably controlled or a precautionary
statement should be included in the procedure

30. Verification of Reference Method
or pharmacopeia method
 The suitability of compendial method must be verified that they
are suitable for the DS/DP analysis in that laboratory conditions
following characteristics should be checked.
 Specificity
 Intermediate precision
 Stability of samples solution (defined if critical)

31. Revalidation consideration
 To Repeat validation of an validated method to
ensure continued compliance with established
requirements after a defined time or in following
situations.
Found that Method performance changes with time
Where some changes were made for improvement in
 Method ,
 Process ,
 DP formulation
 Product etc.
 Degree of validation depends on type of change/ its
criticality

32. Adjustments Vs Changes

33. Other consideration
 When using a non- pharmacopeia method
(justification is needed, comparison with
Pha.method,complete validation required )
 Stress studies should demonstrate that impurities
and degradents from the sample do not interfere with
the quantitation of sample.

34. References
 ICH Q2: validation of analytical procedures, Text and
methodology
 USP: validation of compendial method
 FDA :Analytical procedures and methods validation (Guidance
for industry-draft guidance)
 Emea : recommends to follow ICH Q 2
 WHO :supplementary guidelines on GMP :validation Annexure 2
: Analytical method validation
 Book: validation of analytical method from agilent technologies
 Book: Method validation in pharmaceutical analysis by j.Ermer
and J.H.mill

35. Learning starts from questioning
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36. Learning is a continuous process
For giving this opportunity
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